The present invention relates to a novel heterogeneous immunoassay method for determining the amount of antigens (including ligands and other compounds having antigenic sites or determinants) or antibodies. As is known in the art immunoassays are generally of two types, the so-called separation-free or homogeneous immunoassay, i.e. the method does not require the separation of the unreacted or unbound antibody or antigen from the antigen bound to the antibody, and the so-called separation or heterogeneous immunoassay, i.e. this method requires such a separation step.
In the prior art heterogeneous immunoassay methods, the general procedure is to utilize antigen-antibody bindings with either immobilized antigen or antibody as the means to separate the unbound fraction from the bound fraction. The reaction between the antigen and antibody takes place at a solid-liquid interphase because either the antigen or the antibody is immobilized on a solid material. A marker, which is easy to measure quantitatively because of its activity (e.g. the marker may be an enzyme; a fluorescent molecule which emits light upon excitation by an appropriate light source; a chemiluminescent molecule which will emit light after a chemical reaction such as oxidation; a radioactive molecule; etc.) is linked or bound to either the antibody or the antigen. The marker-antibody or antigen conjugate which precipitates or is insolubilized had been complexed with either the corresponding antibody or antigen. This has more than one disadvantage.
Moreover, in the known heterogeneous immunoassay methods the percentage of marker-antigen or antibody conjugate which is insolubilized, decreases as the concentration of the component (either antigen or antibody) which is being analyzed increases. Therefore, the standard curve when using these methods results in a negative slope.